Invadopodia are actin-based membrane protrusions formed at contact sites between invasive tumor cells and the extracellular matrix with matrix proteolytic activity. Actin regulatory proteins participate in invadopodia formation, whereas matrix degradation requires metalloproteinases (MMPs) targeted to invadopodia. In this study, we show that the vesicle-tethering exocyst complex is required for matrix proteolysis and invasion of breast carcinoma cells. We demonstrate that the exocyst subunits Sec3 and Sec8 interact with the polarity protein IQGAP1 and that this interaction is triggered by active Cdc42 and RhoA, which are essential for matrix degradation. Interaction between IQGAP1 and the exocyst is necessary for invadopodia activity because enhancement of matrix degradation induced by the expression of IQGAP1 is lost upon deletion of the exocyst-binding site. We further show that the exocyst and IQGAP1 are required for the accumulation of cell surface membrane type 1 MMP at invadopodia. Based on these results, we propose that invadopodia function in tumor cells relies on the coordination of cytoskeletal assembly and exocytosis downstream of Rho guanosine triphosphatases.
We have recently identified a tumor suppressor gene TSLC1 on chromosome 11q23.2 by functional complementation of a human lung cancer cell line, A549, through suppression of tumorigenicity in nude mice (1). Furthermore, we have demonstrated the two-hit inactivation of TSLC1 in primary non-small cell lung cancer, hepatocellular carcinoma, and pancreatic cancer, implying its involvement in various human cancers (1). TSLC1 encodes a member of the immunoglobulin superfamily proteins comprising three Ig-like C2-type domains, a single hydrophobic membrane-spanning ␣-helix, and a cytoplasmic domain containing a putative signaling motif (1). From the significant homology of its extracellular domain with those of NCAM1 and NCAM2, 1 we have inferred that TSLC1 is capable of mediating cell-cell interaction.Cell adhesion molecules generally fall into four major classes: the cadherins, the integrins, the selectins, and the Ig superfamily. Among them, Ig superfamily cell adhesion molecules (IgCAMs) are the largest, numbering well over 100 members in vertebrates (2). These well-characterized molecules include NCAMs (3), L1 family CAMs (4), and nectins (5-8). Whereas cadherins and integrins require divalent cations such as Ca 2ϩ or Mg 2ϩ for their adhesive activities (9), IgCAMs are usually Ca 2ϩ -or Mg 2ϩ -independent (10). Moreover, most IgCAMs have preferences for homophilic and/or heterophilic interactions (10). In combination with these interactions, IgCAMs promote a variety of cell-cell associations through cis interaction within the plane of the membranes and/or trans interaction across the membranes (2, 11). For instance, the heterophilic cis interaction between L1 and NCAM appears to enhance the homophilic trans-binding activity of L1 (12, 13). Nectins form cis-homodimers that undergo homophilic and heterophilic trans interactions with each other to mediate cell-cell adhesion (6,11).In this study, the biochemical properties and subcellular localization of TSLC1 were investigated in the cells expressing TSLC1 tagged with GFP or endogenous TSLC1. We report the physiological properties of TSLC1 along with several lines of evidence that TSLC1 is a single transmembrane glycoprotein involved in cell-cell aggregation through homophilic trans interaction.
Renal clear cell carcinoma (RCCC) is a malignant tumor with poor prognosis caused by the high incidence of metastasis to distal organs. Although metastatic RCCC cells frequently show aberrant cytoskeletal organization, the underlying mechanism has not been elucidated. DAL-1/4.1B is an actin-binding protein implicated in the cytoskeleton-associated processes, while its inactivation is frequently observed in lung and breast cancers and meningiomas, suggesting that 4.1B is a potential tumor suppressor. We studied a possible involvement of 4.1B in RCCCs and evaluated it as a clinical indicator. 4.1B protein was detected in the proximal convoluted tubules of human kidney, the presumed cell of origin of RCCC. On the other hand, loss or marked reduction of its expression was observed in 10 of 19 (53%) renal cell carcinoma (RCC) cells and 12 of 19 (63%) surgically resected RCCC by reverse transcription-PCR. Bisulfite sequencing or bisulfite SSCP analyses revealed that the 4.1B promoter was methylated in 9 of 19 (47%) RCC cells and 25 of 55 (45%) surgically resected RCCC, and inversely correlated with 4.1B expression (p < 0.0001). Aberrant methylation appeared to be a relatively early event because more than 40% of the tumors with pT1a showed hypermethylation. Furthermore, 4.1B methylation correlated with a nuclear grade (p 5 0.017) and a recurrence-free survival (p 5 0.0036) and provided an independent prognostic factor (p 5 0.038, relative risk 10.5). These results indicate that the promoter methylation of the 4.1B is one of the most frequent epigenetic alterations in RCCC and could predict the metastatic recurrence of the surgically resected RCCC. ' 2005 Wiley-Liss, Inc.Key words: tumor suppressor gene; bi-sulfite sequencing; two-hit inactivation; recurrence-free survival rate; independent prognostic factor Renal cell carcinoma (RCC) accounts for about 2% of human cancers worldwide, with an incidence of 189,000 and a mortality of 91,000 reported in the year of 2000. 1 Renal clear cell carcinoma (RCCC), which represents 75% of all RCC, exhibits frequent metastasis to distant organs without any clinical symptoms. Furthermore, 40-60% of RCCC tumors without metastasis at first presentation eventually develop metastasis as they progress. 2 Finally, metastatic RCCC becomes refractory to any therapeutic approaches, including chemo-, radio-, and hormonal therapies, resulting in a poor prognosis of patients, with a 5-year survival of less than 10%. 3 Thus, understanding the molecular mechanisms of the development and progression of RCCC is a critical issue for controlling this refractory cancer.Several genetic and epigenetic alterations have been reported in RCCC. The mutation of the VHL gene, associated with loss of heterozygosity (LOH) at the gene locus on chromosomal fragment 3p25-p26, was observed in~50% of sporadic RCCC. 4 Since the VHL encodes a component of an E3 ubiquitin ligase that promotes the degradation of hypoxia-inducible factors, loss of VHL function could be involved in angiogenesis, one of the most characteri...
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